Abstract
Many members of the C-type lectin family of glycan-binding receptors have been ascribed roles in the recognition of microorganisms and serve as key receptors in the innate immune response to pathogens. Other mammalian receptors have become targets through which pathogens enter target cells. These receptor roles have often been documented with binding studies involving individual pairs of receptors and microorganisms. To provide a systematic overview of interactions between microbes and the large complement of C-type lectins, here we developed a lectin array and suitable protocols for labeling of microbes that could be used to probe this array. The array contains C-type lectins from cow, chosen as a model organism of agricultural interest for which the relevant pathogen–receptor interactions have not been previously investigated in detail. Screening with yeast cells and various strains of both Gram-positive and -negative bacteria revealed distinct binding patterns, which in some cases could be explained by binding to lipopolysaccharides or capsular polysaccharides, but in other cases they suggested the presence of novel glycan targets on many of the microorganisms. These results are consistent with interactions previously ascribed to the receptors, but they also highlight binding to additional sugar targets that have not previously been recognized. Our findings indicate that mammalian lectin arrays represent unique discovery tools for identifying both novel ligands and new receptor functions.
Highlights
Zymosan K12E. coli BL21(DE3) O127:H6 O157:H7 K. pneumoniae M. bovis S. aureus MBP-AMBP-CCL-43SP-DCCLo-4n6glutininColK1SP-ACMolML1MRMCRRDCR4D4L+a5ngDerCin-SIGPNrolectiCn DE-nL2dS3oE1C80tinCRD mutASGPARS1GPR2
The design of the lectin array described here includes a number of important technical features associated with the use of biotin tags for immobilization
A key point is the use of simple bacterial expression systems that are easy to generate and give high yields
Summary
Bovine genomic sequences in the National Center for Biotechnology Information databases were screened for annotated instances of the C-type CRD motif. To distinguish sugar-binding proteins from C-type lectin-like domains, the presence of amino acids at positions for ligating to the conserved Ca2ϩ was used to sort the annotated sequences [14]. Most of the tagged CRD fragments were prepared by expression in E. coli, followed by solubilization of inclusion bodies in guanidine and renaturation by dialysis and purification by affinity chromatography on immobilized sugars following protocols developed for many of these proteins from other species This approach ensures that the proteins are correctly folded and have sugar-binding activity. Initial tests of the array were performed using zymosan particles derived from S. cerevisiae because this preparation contains multiple types of sugar structures and binds to most of the C-type CRDs (Fig. 4A).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
